羊膜作为载体培养血管内皮细胞替代自体角膜内皮细胞的研究

Experimental Study on the Corneal Endothelial Cells Substituted by the Vascular Endothelial Cells(VEC) Cultivated on the Amniotic Membrane as its Growth Carrier

目的将羊膜作为血管内皮细胞的生长载体，探讨血管内皮细胞代替自体角膜内皮细胞生理功能的可行性，为大泡性角膜病变的治疗新方法提供实验依据．方法取实验家兔的一侧颈静脉（约3—4cm），用0．25％胰蛋白酶＋0．02％EDTA灌注法收集血管内皮细胞进行原代培养．取新鲜羊膜并去除其表皮细胞，剪成1．5cm×1．5cm大小，表皮面向上平铺于24孔培养板中，再将原代培养的血管内皮细胞传代于羊膜表面，当血管内皮细胞贴壁并长满整个羊膜表面后，我们将载体同血管内皮细胞一起移植到带有大泡性角膜病变的角膜内表面，1～2个月后观察其角膜透明度的变化．结果术后12d左右。实验组和实验对照组（各10只）的角膜水肿及透明度有明显差别．实验组术后4—5d时植片呈白色，高度水肿，通过植片看不见前房，10d后，水肿开始减轻，角膜混浊开始好转，其中7只实验家兔的角膜透过植片能看见前房的组织结构．实验对照组的绝大部分角膜植片水肿加剧，部分角膜植片由于高度水肿而出现坏死溃疡，经统计学分析P〈0．05，两者具有差异性．结论血管内皮细胞具有类似于角膜内皮细胞的生理功能，但其长期的功效有待于进一步的观察和探讨．

Objective Amniotic membrane as the carrier to culture the vascular endothelial cells was investigated in this study in order to explore whether the corneal endothelial cells superseded by the vascular endothelial cells is feasible on the account of directing a kind of the original method to handle the bullous keratopathy. Methods The vascular endothelial cells adhering to the sphagitides lumen of the experimental rabbits were digested and gained by means of the perfusion method with 0.25% tripsin plus 0.02% EDTA. Fresh amniotic membrane without the amniotic epithelial cells was cut into the square tissue about 1.5 cm×1.5 cm and spread evenly in the 24 - well culture dish. Primary cultured cells were subcultured on the amniotic membrane. We would not transplant the vascular endothelial cells feeding on the amniotic membrane until cells is full of the whole amniotic membrane surface. One to two months after the operation, the change of corneal diaphaneity was observed. Results About 12 days since the cell transplantation was performed, the corneal transparency alteration between the experimental groups and the control one is different. The corneal buttons in the experimental group show the severe edema and opacity, and the anterior chamber couldn＇ t be seen unclearly. But, 10 days after the operation, the corneal oedema which begins to extenuate was judged through the indicatrix that the corneal edema in the experimental group has been recovering slowly, among which the anterior chamber tissue of 7 animals was visible through the implant. The corneal edema in the control groups intensified evidently, even, the part of these appeared the ulcerous necrosis as result of the corneal severely edema. There is the existence of difference between two groups （P 〈0. 05） . Conclusions The research supported this conclusion that the vascular endothelial cell has possession of the physiologic property similar to that of the corneal endothelium. But, the long - term effect that the vascular endotheliocyte substitutes the autogenous endothelium corneal will be explored and observed further.